Figure 4.

Western blot analysis of S100A9 interactions with ApoE isoforms during the amyloid formation. (A) SDS-PAGE of S100A9 without and with ApoE isoforms after 70 h incubation at 42 °C. Lines of SDS-PAGE indicated from left to right: S100A9 alone; S100A9 with ApoE3-DOPC; S100A9 with ApoE3; S100A9 with ApoE4-DOPC; S100A9 with ApoE4; ApoE3-DOPC alone; ApoE3 alone; ApoE4-DOPC and ApoE4 alone. (B) Western blot corresponding to SDS-PAGE performed using anti-S100A9 polyclonal antibody (Invitrogen, Carlsbad, CA, USA). As indicated from left to right above the Western blot: S100A9 alone; S100A9 with ApoE3-DOPC; S100A9 with ApoE3; S100A9 with ApoE4-DOPC and S100A9 with ApoE4. Bands of ~50 kDa marked in the Western blot correspond to the complexes of S100A9 with ApoE3 or ApoE4, respectively. Other bands corresponding to S100A9 and S100A9-ApoE complexes are marked on the right side from the blot. Totals of 50 μM S100A9 and 2 μM of corresponding ApoE isoforms in PBS buffer were incubated at 42 °C for 70 h. (C) Size exclusion chromatogram of the supernatants of S100A9 aggregated alone for 70 h (black line) and S100A9 aggregated in the presence of ApoE4 (red line) and subjected to centrifugation. The numbers of the chromatographic peaks correspond to the lane numbers in the following Western blot. (D) Western blot with anti-S100A9 polyclonal antibodies (Santa Cruz) of the pellets and the various peaks of supernatant, collected during elution of S100A9-aggregated samples through size exclusion chromatography. Totals of 100 μM S100A9 and 5 μM ApoE4 were incubated in PBS buffer at 42 °C for 70 h. The band with ~50 kDa molecular weight characteristic of the complex of S100A9 with ApoE4 isoform in the pellet and void volume (Lane 6) can be seen. Other bands corresponding to S100A9 and S100A9–ApoE complexes are marked on the right side of the blot.